DESCRIPTION: Cytokinesis is an essential process required for the separation of daughter cells at the completion of cell division. Dr. Sullivan takes the unusual approach of studying cytokinesis through the examination of the model system of metaphase furrow formation in the Drosophila syncytial blastoderm. During metaphase of each of the cell cycles that occur after the embryonic nuclei have migrated to the cortex, but prior to cellularization, invaginations of the plasma membrane occur that form furrows that extend into the egg. As Dr. Sullivan has elegantly shown, at least one of the function of this furrow is to separate adjacent spindles, which would otherwise fuse together. Metaphase furrow formation and cytokinesis share several properties, of which the most striking is the common utilization of several molecular components. By screening collections of maternal effect lethal mutations, Dr. Sullivan's lab has identified a series of genes that affect the elaboration of the metaphase furrow. This application is primarily directed to investigating two of these: nuclear fallout (nuf), and grapes. The current understanding of the function of these genes is as follows. grapes encodes a protein in the serine/threonine kinase family that is found in the nucleus except during prometaphase-interphase of the cell cycle. Dr. Sullivan hypothesizes that this protein is sequestered from potential targets within the nucleus, but then phosphorylates targets in the cytoplasm needed for metaphase furrow formation. The function of grapes is not required for the recruitment of anillin (which probably helps form a scaffold for contractile ring) to the metaphase furrow. However, the level of actin in the furrows is reduced, and myosin fails to localize there. nuf encodes a protein with domains predicted to form coiled-coils. Preliminary analysis with rhodamine-conjugated bacterially-synthesized nuf protein injected into embryos suggests that this protein localizes to the metaphase furrows. Again, in nuf mutant embryos anillin localizes properly to the metaphase furrows, but the recruitment of actin and myosin to these structures is abolished. Dr. Sullivan speculates that the nuf protein stabilizes, assembles, or delivers components to the metaphase furrow. The first specific aim of the proposal concerns further phenotypic characterization of the grapes and nuf mutants. Actin and myosin dynamics in mutant embryos will be followed in detail by recording living embryos injected with fluorescently-labelled actin, and by staining fixed embryos with anti-myosin. These experiments will help define the cellular basis for the metaphase furrow defects observed. The second specific aim is devoted to understanding the cellular localization of grapes and nuf proteins in greater detail. Antibodies against the nuf protein are presently being prepared; this analysis will be supplemented by further investigations with injected rhodamine-labelled bacterially-synthesizednuf protein and with GFP/Nuf fusion constructs to be made. Experiments will be performed to determine the distribution of nuf relative to other structures and components (such as actin, tubulin, centrosomes) that behave in a dynamic fashion during the blastoderm divisions. Similar experiments will be performed with antibody already generated against the grapes protein. Of interest here will be determination of the timing of grapes redistribution between nucleus and cytoplasm compared with nuclear envelope breakdown and reformation as assayed with anti-lamin antibodies and other techniques. The distribution of grapes protein will also be examined in embryos treated with drugs that disrupt the actin or tubulin cytoskeletons. A third line of investigation concerns the genetic dissection of metaphase furrow formation using the mutations and antibody reagents generated in the Sullivan laboratory. Currently seven, and perhaps as many as nine genes needed for proper behavior of the metaphase furrow have been identified. A number of antibodies against component of the furrow are available; these include antibodies against the actin, myosin, septins, anillin, spectrin, and cofilin. The pairing of the panel of mutations and antibodies will allow the determination of dependency pathways in the assembly of the metaphase furrow. The fourth specific aim is to identify and characterize the proteins that interact with nuf and grapes products. Dr. Sullivan will explore both biochemical and genetic approaches. He will use sucrose gradient fractionation of embryonic extracts to determine whether these proteins sediment as members of multimeric complexes. Affinity chromatography of these extracts will be performed on columns either with bound, affinity purified anti-nuf or anti-grapes antibodies, or with bound, bacterially-synthesized fusion proteins. Proteins that interact with these columns will be made in preparative amounts, and used as antigens to generate rabbit polyclonal antibodies. The intracellular distribution of these proteins will be used for immunofluorescence studies. The corresponding genes will be cloned by screening cDNA expression libraries with the antibodies. Additional experiments to determine whether the nuf protein, which appears to localize to the cleavage furrows, can associate with F-actin. Finally, because the grapes protein appears to be a kinase, efforts will be made to characterize the grapes kinase activity. Potential targets including myosin will be tested. If substrates are found, the level of grapes kinase activity as a function of the cell cycle will be determined using extracts from individually staged embryos. Efforts will be made to determine whether grapes itself is phosphorylated, particularly by the polo kinase, mutations in which yield phenotypes similar to grapes. The genetic approaches to be employed include a screen for suppressors of the female sterility associated with grapes or nuf, and a screen for enhancers that display second site non-complementation with these mutants.